Prostate cancer is the most common non-cutaneous cancer and the second leading cause of cancer death in American men. Recent recommendations by the US Preventative Services Task Force on PSA screening highlighted the public health concerns relating to the imprecision of PSA testing and the overtreatment of prostate cancer. PSA's limited ability to discriminate cancer from benign disease leads to well over a million prostate biopsies each year in the United States. Moreover, low grade prostate cancer is often non-fatal and the excessive use of definitive therapies such as surgery has led to significant morbidity and costs. The long-term objective of this project is to develop novel panels combining the TMPRSS2:ERG fusion with long noncoding RNAs (IncRNAs) that will allow improved clinical risk assessments for prostate biopsies and active surveillance of low grade cancer. We will employ transcriptome sequencing (RNA-Seq) in Aim 1 on a large panel of prostate cancer tissues and cell lines to provide an unbiased assessment of RNA species whose aberrant expression associates with prostate cancer or prostate cancer disease progression. This innovative approach is able to capture all disease-associated RNAs, including mRNAs encoding for proteins and long non-coding RNAs (IncRNAs). Sequencing data is processed to distinguish high-confidence, recurrent transcripts from background or spurious sequencing signal. High-confidence transcripts are then further categorized relative to annotated genes. From the final compendium of prostate cancer transcripts, we will then nominate and validate transcripts dysregulated in prostate cancer. We will select the most promising transcripts for assessment and validation in urine as putative prostate cancer biomarkers. Taking advantage of the large clinical data and tissue repositories in Aim 2, novel IncRNAs will be multiplexed with other existing RNA-based urine tests, including assays for the TMRPSS2:ERG RNA and the known prostate cancer IncRNA PCA3, to develop a panel assay with a sufficiently high negative predictive value to prevent unnecessary prostate biopsies. Aim 3 will similarly deploy ncRNAs to develop a panel capable of identifying high grade cancer based on a urinary assay. Our latest preliminary data suggests that PCAT-114 (SChLAP-1) is our most promising IncRNA for clinical development as it is associated with aggressive prostate cancer in tissues and in urine.